We are studying the human virome in children with fever. For the purposes of these studies, the human virome is defined as the total collection of viruses infecting humans, including viruses causing acute, persistent, or latent infection, but not including bacteriophages or viruses integrated into the human genome without evidence of replicative capacity. Children with fever are frequently infected with viruses and thus are very suitable for early studies of the human virome. In this project, previous studies on normal children with and without fever will be extended to include immunocompromised children. The reasons for studying immunocompromised children are 1) to increase our knowledge of the viruses causing fever in immunocompromised children to improve their medical management, 2) to learn about the influence of the immune system on the composition of the human virome, and 3) to search for novel viruses that might be present in these children because of their immunocompromised state. A series of studies will also be performed to improve the system we have already established for using high throughput nucleotide sequencing to define the human virome. Thus, this project will enroll 400 children who are immunocompromised for varying reasons including receipt of a hematopoietic stem cell or solid organ transplant, presence of a congenital immunodeficiency, HIV/AIDS, and treatment of diseases with medications that suppress the immune system. Samples of blood, nasopharyngeal secretions, and stool will be collected from 200 children on a single occasion to survey the viruses that are present. In addition, the same set of samples will also be collected from 100 children having episodes of fever, at the time of the febrile episode and 1 and 6 months later. Finally, in order to study directly the effects of the immune system on the virome, the same set of samples will be collected from 100 children who are undergoing transplantation before the transplant and 1 and 6 months later. Samples will be analyzed using comprehensive panels of virus-specific PCR assays and by next generation high throughput nucleotide sequencing. In order to refine the established pipeline for detecting viruses by metagenomic sequencing, a series of new computational methods to detect known and novel viruses will be investigated. New platforms for high throughput sequencing that may allow longer sequencing reads or increased numbers of reads will also be evaluated. Finally investigations of novel viruses detected will be carried out, including whole genome sequencing and investigations of their prevalence and clinical manifestations using virus-specific PCR assays designed based on the nucleotide sequence. This project is expected to provide medically useful information about the range of viruses producing illness in immunocompromised children and should greatly improve methods for studying the human virome. PUBLIC HEALTH RELEVANCE: This project will use the most advanced molecular techniques to define the full range of viruses present in children whose immune systems are not normal, both when they are having episodes of fever and when they are medically stable. It will also improve the ability to use the most modern and powerful high throughput nucleotide sequencing technology for the detection of viruses in human samples. This enhanced capability will be broadly applicable to studies seeking to define the range of viruses associated with human health and disease.